How mammalian neural circuits are assembled during development remains a largely unanswered question. We recently identified a mechanism restricting neural circuit assembly, a process we determined is mediated by Nogo receptor signaling via the GTPase RhoA1. We propose to define the principal characteristics of this synapse restriction mechanism in this study. A major obstacle limiting our understanding of the neural circuit assembly process has been the inability to visualize the synaptic wiring process live while simultaneously recording the activity of proteins regulating synaptogenesis. Here, we demonstrate we have overcome these obstacles. Combining state-of-the-art live imaging microscopy with multiple labeling approaches in concert with the introduction of fluorescent sensors in axons and dendrites undergoing synapse development, we can covisualize signaling molecules that we propose restrict (RhoA) or promote synapse assembly (calcium). We hypothesize that NgRs block axo-filopodial contact formation by RhoA-mediated inhibition of postsynaptic calcium signaling. Importantly, our preliminary studies reveal we can detect distinct RhoA and calcium signals in dendrites and axons, observe changes in our sensors during axo-dendritic contact and track the fidelity and strength of these contacts over time. These findings demonstrate we are positioned to address our central hypothesis. We aim to Define RhoA's Role in Restricting Synapse Assembly (Aim 1), determining whether focal activation or global inhibition of RhoA alters synaptic contact stability and then examine the effects of NgR family loss on synaptic contact fidelity (Aim 3). Our preliminary work also reveals that RhoA activity peaks at short-lived but not stable synaptic contacts, suggesting RhoA may inhibit contact stabilization. Further, NgR loss profoundly calcium and RhoA signaling at synaptic contact sites. These findings strongly support our proposed hypothesis. The experiments in this proposal will give us an unprecedented view of the signals underlying synapse assembly and the role the NgR family plays in controlling them. These studies will provide a roadmap for defining the events and signals driving synapse assembly. A roadmap we plan to build on in the future using other sensors and assembly molecules to define how numerous circuits are assembled during development and disrupted in disease.